Neurovascular units (NVU) are the structural and functional elements of the brain and are exquisitely susceptible to ischemic stroke. Stroke is the third leading cause of death and the leading cause of long-term disability, posing an enormous social and economic burden to society. Although the FDA has approved tissue plasminogen activator for restoring blood flow, no neuroprotective medicine is available for stroke victims. Ischemic preconditioning (IPC) has recently been shown to be able to induce ischemic tolerance of the brain against subsequent ischemic injury to the NVU; however, the underlying protective mechanisms are not clear. Using both in vitro and in vivo methods, we have obtained exciting preliminary results suggesting that IPC protects against ischemic injury via the transcription factor Nrf2 and the generation of endogenous lipid electrophiles that create mild oxidative stress. Suppressing Nrf2 or neutralizing electrophiles with N- acetylcysteine eliminates the ischemic tolerance. Electrophiles robustly activate Nrf2, induce phase 2 enzymes, and protect mouse brains, cultured rat primary cortical neurons, mouse brain microvessel endothelial cells (MBMEC), and astrocyte-endothelial cell co-cultures from ischemic injury. Furthermore, treating MBMEC cells with 4-HNE or preconditioning attenuates blood-brain barrier (BBB) damage induced by ischemia. The purpose of this proposal is to further investigate the mechanisms responsible for Nrf2 activation and neurovascular protection induced by IPC. The overall hypotheses are that IPC causes mild oxidative stress in the brain, leading to the generation of sublethal levels of lipid electrophiles; these electrophiles then activate the Nrf2 pathway by suppressing both Keap1 and GSK3, thereby protecting NVU components. Three specific aims are proposed: Aim 1 tests the hypotheses that IPC offers long-term neuroprotection against focal ischemia in mice and that Nrf2 and electrophiles are required for the sustained protection; Aim 2 tests the hypothesis that lipid electrophiles activate Nrf2 by inhibiting Keap1 and GSK3 in NVU component cells; Aim 3 tests the hypothesis that IPC reinforces BBB via upreglulating CDH5 and that Nrf2 action protects the BBB and NVU in ischemic brain. This thorough investigation of the protective mechanisms of IPC may help develop future therapies that boost endogenous regulatory mechanisms in stroke victims.